PROJECT SUMMARY/ABSTRACT New tools to selectively regulate neurons have revolutionized causal experimentation. Optogenetics provides an array of elements for specific biophysical control, while designer chemogenetic receptors provide a minimally invasive method to control circuits in vivo by peripheral injection. We have developed a strategy for selective regulation of activity in specific cells that integrates opto- and chemo-genetic approaches, and thus allows manipulation of neuronal activity over a range of spatial and temporal scales in the same experimental animal. Light-sensing molecules (opsins) are activated by biologically produced light through luciferases upon peripheral injection of a small molecule, which crosses the blood-brain barrier. Such BioLuminescence-driven OptoGenetics (?BL-OG?) is a minimally invasive method like chemogenetics, but one that leverages the full array of bioluminescent and optogenetic options. Importantly, BL-OG allows conventional fiber optic activation while at the same time providing chemogenetic access to the same sensors. This opens, in principle, the entire optogenetic toolbox for complementation by a chemogenetic dimension. Further, because different forms of luciferases use non-cross reactive luciferins, multiple distinct effects can be independently and conjointly controlled in the same animal. We demonstrated proof of concept for this technology by using fusion proteins that directly link Gaussia luciferase (GLuc) to opsins, creating luminescent opsins (luminopsin, LMO). Here, we describe our next steps to increase the benefit of this technology for the field. We will expand the range of BL-OG options, increase their potency, and systematically quantify BL-OG impact in vitro and in vivo. In Aim I, we will generate new luciferases with increased light emission and luciferase/luciferin pairs with non- overlapping substrates to allow multiplexing. In Aim II, we will develop an extended toolkit of luciferase-opsin combinations and test their efficacy in vitro. In Aim III, we will validate and quantify the efficacy of bioluminescence activation of neural circuits in vivo by and directly compare stimulation of LMOs versus fiber optics versus DREADDs. Reflecting the basic science and clinical importance of BL-OG and the expertise of the investigators, we will use defined networks in neocortex and thalamus targeted with viral vectors expressing activating and silencing LMOs and DREADDs. The overall outcome of our work will be the optimization and validation of a novel, highly flexible tool set for bimodal optogenetic and chemogenetic interrogation of neuronal circuits in living animals. The proposed work will give the neuroscience community new molecules and comparative data to aid in making an informed decision when choosing among the various tools that may meet their specific experimental needs.